BIOSYNTHESIS of Taxol

紫杉醇的生物合成

• 批准号: 7802959
• 负责人: RODNEY B CROTEAU
• 金额: $ 32.07万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7802959
• 关键词: 14p; Acetyltransferase; Anabolism; Antineoplastic Agents; Biological; Biological Process; Cell Culture Techniques; Cells; Cloning; Coenzyme A; Complementary DNA; Complex; Cultured Cells; Diterpenes; Engineering; Enzyme Gene; Enzymes; Foundations; Future; Generations; Genes; Goals; Lead; Ligase; Metabolic; Methods; Mixed Function Oxygenases; Modification; Molecular Genetics; Oxidases; Paclitaxel; Pathway interactions; Pharmaceutical Preparations; Production; Reaction; Route; Side; System; Taxoids; Taxus; Transgenic Organisms; base; cost; drug production; enzyme biosynthesis; epoxidase; expression cloning; feeding; genetic manipulation; geranylgeranyl diphosphate; improved; in vivo; overexpression; oxetane; phenylisoserine; tool

Total synthesis of the anticancer agent Taxol is not practical and, for the foreseeable future, the supply of this diterpenoid drug, and its precursors for semisynthesis, must continue to rely on biological processes involving the isolation from yew (Taxus) species or cell cultures derived therefrom. Improvement of these biological methods of production must be based upon a detailed understanding of the complex pathway for Taxol biosynthesis, the enzymes which catalyze the sequence of reactions, and the genes for these enzymes, especially those responsible for slow steps, since the molecular genetic manipulation of the pathway can be expected to lead the production of the drug in high yield at more reasonable cost. The goal of improving Taxol production will be reached by determining the types and order of enzymatic steps from the diterpenoid branch-point intermediate geranylgeranyl diphosphate to the end- product, by cDNA cloning of the responsible genes, and by assessing the flux contribution of each step (and of diversionary side routes) by gene overexpression and suppression. Defining this multistep pathway is accomplished through the use of cell-free enzyme systems from induced yew (Taxus) cultured cells, combined with in vivo feeding studies, to determine the progression from simple to complex metabolites. This systematic approach has identified several early, intermediate and late steps of the Taxol pathway, and provided the tools for cDNA isolation with which thirteen pathway genes have been obtained and characterized by a broad range of cloning and expression strategies. The specific aims of this project are: 1. to clone and characterize the remaining five genes corresponding to intermediate enzymatic steps that complete modification of the taxoid core (Clp-hydroxylase, C9a-hydroxylase and C9 oxidase, C4,C20-epoxidase and oxomutase catalyzing formation the oxetane ring); 2. to clone and characterize the remaining two genes corresponding to the aroyl CoA ligase and C2'-hydroxylase needed to complete assembly of the C13 Af-benzoyl phenylisoserine side-chain; 3. to clone the corresponding genes and characterize the taxoid C7-0-, C9-O-, and C13-0-acetyltransferases which, along with taxoid 14p-hydroxylase, constitute major diversions of intermediate taxoids away from Taxol; and 4. to engineer Taxus cells for overexpression and suppression of each pathway (and side-route) gene, and to assess the influence on metabolic profile and yield as a means of ordering the sequence of reactions and defining flux controls at each step. Completion of these objectives will provide an understanding of Taxol biosynthesis and the foundation for multigene transgenic approaches to improve theproduction of Taxol, and of related intermediates for semisynthesis of Taxol and second generation taxoid drugs.

抗癌剂紫杉醇的完全合成是不切实际的，在可预见的将来，它的供应 二萜药物及其半合成的前体必须继续依靠涉及的生物学过程 从紫杉（出租车）物种或细胞培养物中分离出来。这些生物学方法的改进 生产必须基于对紫杉醇生物合成复杂途径的详细理解，即酶 这会催化反应序列，以及这些酶的基因，尤其是负责缓慢步骤的酶的基因， 由于可以预期该途径的分子遗传操纵将导致该药物的产生高 以更合理的成本产量。通过确定类型和 从二萜的分支点中间的黄烷基凝酰二磷酸到末端的酶促步骤的顺序 产物，通过负责基因的cDNA克隆，并通过评估每个步骤的通量贡献（和 通过基因过表达和抑制作用的转移侧路线）。定义此多步途径已完成 通过使用来自诱导紫杉（Taxus）培养细胞的无细胞酶系统，并结合体内进食 研究，以确定从简单到复杂代谢产物的发展。这种系统的方法已经确定 紫杉醇途径的几个早期，中间和晚期，并提供了cDNA隔离的工具 已经获得了13个途径基因，并以广泛的克隆和表达策略为特征。 该项目的具体目的是：1。克隆并表征其余的五个基因 中间酶促步骤完成了符号核（CLP-羟化酶，C9A-羟化酶和 C9氧化酶，C4，C20-环氧酶和Oxomutase催化酶形成氧烷环）； 2。克隆并描述 剩下的两个基因对应于Aroyl CoA连接酶和C2'-羟化酶，需要完成组装的组装 C13 Af-苯甲酰苯二糖苷的侧链； 3。克隆相应的基因并表征符号C7-0-- C9-O-和C13-0-乙酰基转移酶，与Taxoid 14p-羟化酶一起构成了主要转移 远离紫杉醇的中级紫类动物； 4。要设计出出租车细胞的过表达和抑制 途径（和侧面路由）基因，并评估对代谢概况和产量的影响作为订购的手段 每个步骤的反应序列和定义通量控制。这些目标的完成将提供 了解紫杉醇生物合成和多基因转基因方法的基础以改善生产 紫杉醇和相关中间体的半合成和第二代类囊素药物的半合成。